Summary and Background: Auditory and vestibular systems are required for proper hearing and balance. These systems require sensory hair cells of the inner ear, to sense sound and process vestibular stimuli. To reliably transmit auditory and vestibular information, hair cells use specialized ribbon synapses. Our studies combine genetic, molecular, and imaging-based approaches to identify the structural and functional processes underlying synapse formation and function in hair cells. For our studies we use the zebrafish lateral-line system in order to study hair-cell development and function, in a live, transparent preparation. We have an extremely powerful collection of transgenic zebrafish that label synaptic structures to either assess synaptic morphology or function using genetically encoded fluorescent proteins. We are combining these microscopy-based approaches with CRISPR technology to create mutant zebrafish in order to identify genes required for synapse formation, function and regeneration. With this knowledge we aim to apply our understanding of these processes in order to understand how to properly reform hair cells and synaptic structures when they are loss or damaged after hearing loss. This report summarizes the fifth year for the Section on Sensory Cell Development and Function. Our main focus has been publishing research projects initiated at the NIH, and exploring ways to expand upon and initiate new research. The lab continues to demonstrate proficiency in several areas: animal husbandry, the creation of mutant and transgenic zebrafish, electrophysiology, function-based confocal imaging and applying new microscopy-based approached to our system. General accomplishments for this fiscal year include moving our zebrafish colony from the NIH zebrafish facility in 6 to the new NIDCD facility in building 35A. In addition, we several ongoing collaborations with other labs at the NIDCD, and the surrounding area. Through these collaborations we will apply our expertise and tools in zebrafish to advance and complement other aspects of auditory research. Projects in the lab: 1) Understanding the hair-cell synapse formation and maintenance Determining molecules required for ribbon synapse formation Very little is known regarding the molecules required for forming ribbon synapse. Based on a RNAseq data set we are creating mutant zebrafish to test the role of candidate adhesion molecules in hair-cell synapse formation. Overall, our findings from this work support that molecules required to form synapses in hair cells are also required at retinal ribbon synapses. Our data provides fundamental insight into ribbon synapse formation that will help us understand how reform these structures after hair cell or synapse loss. How does hair-cell activity shapes ribbon synapse formation and maintenance Previous research has indicated that mutants with impaired presynaptic calcium activity in the hair cell fail to maintain synaptic integrity throughout development. We aims to characterize the specific elements and molecular components of activity in the hair cell necessary for the formation and stabilization of mature, functional ribbon synapses. We have examined the requirements of the hair-cell proteins Pcdh15, CaV1.3, Vglut3 and Otoferlin on synapse formation and maintenance. Respectively, these proteins have been shown to mediate mechanotransduction, presynaptic calcium influx, glutamate transmission and vesicle fusion. Our results indicate that vesicle fusion, but not glutamate transmission, is critical to maintain hair-cell synapses. Currently we are focused on understanding the contents in synaptic vesicle that are important to maintain hair-cell syanpses. How does presynaptic calcium activity alter hair-cell synapse assembly Our current understanding of hair-cell synapse formation has been largely obtained by studies that examined morphological or functional changes at single time points during development. Although this work has been informative, development is dynamic, and local synaptic activity is thought to play an active role shaping synapse assembly. We have used the zebrafish model to examine the relationship between synaptic activity and ribbon assembly in vivo. Our previous findings indicated that a pharmacological activation or inhibition of synaptic calcium could push developing synapses towards assembly or disassembly, respectively, on relatively short timescales. Currently we have examined how presynaptic calcium activity controls ribbon development. Our work indicate that presynaptic calcium load calcium into synaptic mitochondria. Mitochondrial calcium loading can alter the metabolic redox state of the hair cell by altering NAD+/NADH levels. Ribeye, the main component of ribbon synapses contains a NAD(H) binding domain that can impact Ribeye-Ribeye self-assembly and ribbon formation. NAD+ and NADH, act directly to increase and decrease ribbon formation during hair-cell development. This work provides mechanistic insight into how activity in hair cells regulates synapse assembly during development and represents important knowledge required to reform synapses under pathological conditions. 1) Determine how a sensory stimulus are encoded within an intact sensory system Synaptic integration among populations of sensory hair cells Analysis of auditory transduction among ensembles of sensory cells in the mammalian inner ear in vivo is challenging due to their location deep within the temporal bone. To overcome this limitation, we used optical indicators to investigate mechanosensation and transmission among collections of sensory hair cells in intact zebrafish. Our imaging reveals a previously undiscovered disconnect between hair-cell mechanosensation and synaptic transmission. We show that suprathreshold mechanical stimuli able to open mechanically-gated channels, are unexpectedly insufficient to evoke vesicle fusion in the majority of hair cells. Overall, this work has important implications for inner-ear recovery after hair-cell loss, where it is important to understand how many hair cells must be regenerated, in order to translate sensory stimuli into meaningful behavior. We are currently investigated the mechanism underlying sensory integration and synaptic facilitation among populations of hair cells in vivo.